The key role and important value of β-glucanase has been revealed not only in biomass conversion but also in various industries,including food processing,textiles,laundry detergents,and paper manufacturing.A thermostable β-glucanase with high catalytic activity is a particularly attractive candidate for complete degradation of cellulose and brewery.In this study,the directed evolution was employed to increase the activity of β-glucanase from Thermotoga maritime by error-prone PCR.The cel12B gene encoding β-glucanase in recombinant vector pET-20b-Cel12B was amplified by error-prone PCR at a definite concentration of Mn2+,different concentration of Mg2+ was used to optimize the PCR for enhancing specificity and mutation,and PCR products were cloned into pET-20b to form recombinant plasmid pET-20b-Mu-Cel12B,followed by generating a mutant molecular library in E.coli.The mutant variants of the encoded-glucanase with enhanced activity were selected by the change method of Congo Red staining.Three mutants(Mut1,Mut2 and Mut3) that showed increased enzyme activity compared to the wild type were selected according to the size of transparent circles on Congo Red plates.After the three mutants were induced under the same conditions as wild enzyme,the results showed the enzyme activity of Mut1,Mut2,Mut3 was 2.1-fold,3.2-fold,and 3.7-fold than that of wild-glucanase,respectively.